ProjectSummary/Abstract This research program will establish the fundamental chemical principles underlying the newly discovered Mn/Fe proteins. The active sites of these proteins defy conventional inorganic wisdom to spontaneously assemble a bimetallic cofactor that contains two different transition metals in nearly identical coordination environments. Following assembly, oxygen is activated across the metal centers to induce a one- or two- electron oxidation reaction, with regeneration occurring via intermolecular electron transfer. Unlike the well- studied diiron enzyme homologs, the molecular-level details of these processes in Mn/Fe proteins remain unknown. Because Mn/Fe-containing proteins have been identified primarily in extremophilic and pathogenic organisms, including many species of Chlamydia and Mycobacteria, it has been suggested that the heterobimetallic cofactor may offer resistance against reactive nitrogen and/or oxygen species generated by thehostimmunesystem.TheproposedstudieswillprobethishypothesisusingtheR2loxproteinsasamodel scaffold, examining reactivity of the Mn/Fe cofactor relative to a diiron site. Initial studies by the PI have indicated aerobic assembly of R2lox proceeds through two distinct intermediates, identified by time-resolved optical and EPR spectroscopy. The proposed work will use an array of spectroscopic techniques, including optical, resonance Raman, CW- and pulsed EPR, and Mssbauer, to elucidate the electronic and geometric structures of these intermediates. Targeted mutagenesis around the active site will allow identification of key residues responsible for selective metal binding, ultimately revealing the mechanism by which assembly and activation proceed. To gain a comprehensive picture of the processes occurring at the active site, the redox properties of Mn/Fe cofactors will be characterized to determine the thermodynamics and kinetics of electron transfer,anecessarycomponentforefficientcatalysis.Finally,thescopeofreactivityofMn/Feproteinswillbe expanded using protein engineering techniques. Rational metalloprotein design will be coupled with directed evolutionapproachestogeneratehighlyactiveenzymescapableofselectiveoxidationoftargetedsubstrates. Collectively, the proposed research program will fill many existing knowledge gaps about the Mn/Fe proteins, better resolving the physiological role that these unique cofactors may play in the metallobiochemistry of microbes.